Packing materials for liquid chromatography (LC) are generally classified into two types: organic materials, e.g., polydivinylbenzene; and inorganic materials typified by silica. Many organic materials are chemically stable against strongly alkaline and strongly acidic mobile phases, allowing flexibility in the choice of mobile phase pH. However, organic chromatographic materials generally result in columns with low efficiency, leading to inadequate separation performance, particularly with low molecular-weight analytes. Furthermore, many organic chromatographic materials shrink and swell when the composition of the mobile phase is changed. In addition, most organic chromatographic materials do not have the mechanical strength of typical chromatographic silicas.
Due in large part to these limitations, silica is the material most widely used in High Performance Liquid Chromatography (HPLC). The most common applications employ silica that has been surface-derivatized with an organic group such as octadecyl (C18), octyl (C8), phenyl, amino, cyano, etc. As stationary phases for HPLC, these packing materials result in columns that have high efficiency and do not show evidence of shrinking or swelling.
However, a further problem associated with silica particles and polymer particles is packed bed stability. Chromatography columns packed with spherical particles can be considered to be random close packed lattices in which the interstices between the particles form a continuous network from the column inlet to the column outlet. This network forms the interstitial volume of the packed bed, and acts as a conduit for fluid to flow through the packed column. In order to achieve maximum packed bed stability, the particles must be tightly packed, and hence, the interstitial volume is limited in the column. As a result, such tightly packed columns afford high column backpressures that are not desirable. Moreover, bed stability problems for these chromatography columns are still typically observed because of particle rearrangements.
A trend in current HPLC development is the miniaturization of column diameters that is driven by the often limited amount of samples originating from such areas as the life sciences. For mini- and microbore columns as well as capillary columns, the trade-off between particle size and backpressure becomes even more pronounced. For example, MacNair et al.(1) required specifically designed hardware that enabled an operating pressure as high as 500 MPa in order to achieve a HPLC separation of a tryptic digest in a 25 cm long capillary column packed with 1 μm silica particles. The pressure is one order of magnitude higher than the typical 40 MPa limitation of a commercial HPLC system.
In an attempt to overcome the combined problems of packed bed stability and high efficiency separations at low backpressures and high flow rates, several groups have reported the use of monolith materials in chromatographic separations. Monolith materials are characterized by a continuous, interconnected pore structure of large macropores, the size of which can be changed independent of the skeleton size without causing bed instability. The presence of large macropores allows liquid to flow directly through with very little resistance resulting in very low backpressures even at high flow rates.
Monolith columns have been designed to disobey the trade-off rule associated with packed particle beds. Theoretically, they can exhibit combined properties of low backpressure and high efficiency that go beyond the limits of packed particle columns in pressure-driven liquid chromatography. Capillary monolith columns comprising polymeric, inorganic silica and organic-inorganic hybrid materials have been studied and reported in the literature.(2,3) The polymeric monoliths are made primarily via a radical polymerization of methacrylate or styrene-divinylbenzene(DVB) monomers and are used under electroosmotic flow in electrochromatography applications and low pressure pump driven applications because of their limited mechanical strength under high pressure.
Silica monoliths have also been applied in HPLC separations by Nakanishi et al.(3) and have demonstrated an efficiency similar to 5 μm particles but with permeability 25-30 times higher. However, due to the shrinkage of the silica skeleton, silica capillaries with an I.D. larger than 50 μm showed much lower efficiency, and in all cases 5-15% of the length of each capillary end had to be cut off to remove large voids caused by shrinkage that formed between the monolith and capillary wall before the capillary could be used.
In another publication, Nakanishi at al.(4) demonstrated the possibility of making a capillary column of 200 μm internal diameter (I.D.) from a mixture of tetramethoxysilane and methyltrimethoxysilane. However, these hybrid-type silica monoliths capillaries still had large voids caused by shrinkage that formed between the monolith and capillary wall and required cutting of 5-15% of the length of each capillary end before use. The hybrid-type silica monolith also had a three-fold increase in separation impedance versus the analogous silica monolith column of 50 μm I.D.
Polymeric capillary monolith columns prepared by a UV polymerization of (3-methacryloxypropyl)trimethoxysilane were first reported by Zare et al. in 2001.(5) The elution order of the Zare column is similar to that of a reversed-phase column where larger molecular weight or more hydrophobic analytes elute later than the smaller molecular weight or more hydrophilic analytes, indicating that the polymerized methylacrylate groups are located on the surface of the monolith structure. Although Zare's work has been successfully applied in electrochromatography, poor column efficiency, poor adhesion between the capillary wall and the monolith structure, and inhomogeneity of the monolith structure were observed in pressure driven separations.(6) Moreover, as a consequence of the utilization of photopolymerization rather than thermal polymerization, the polyimide coating of the glass capillary must be removed prior to use. This unprotected fused silica tubing becomes very fragile and is easily broken. Therefore, only columns with a limited length can be prepared by this method.
Current monolith columns have significant shrinkage, resulting in poor wall adhesion, and consequently, only columns with an I.D. of less than 150 μm have been prepared. Therefore, a need exists for novel materials that overcome the problems that are associated with known materials. In particular, there is a need for monolith materials with increased resistance to shrinkage and enhanced wall adhesion that can be used to prepare chromatographic columns with an I.D. of 150 μm and greater.